Background: Leptospiral infections in poikilothermic (cold blooded) animals have received very little attention and the literature concerning natural infections of these animals is limited. The aim of this study was to determine the prevalence of leptospiral antibodies in reptiles, imported into Slovenia and intended to be pets in close contact with humans. A total of 297 reptiles (22 snakes, 210 lizards and 65 turtles) were tested for specific antibodies against serovars of Leptospira interrogans sensu stricto using the microscopic agglutination test (MAT). Live cultures of different serovars were used as antigens. MAT was performed according to standard procedures and the degree of reaction was interpreted by estimating the percentage of agglutinated leptospires. Samples showing titres of ≥ 50 against one or more serovars were considered as positive. Results: Antibodies against seven pathogenic serovars of L. interrogans sensu stricto were detected in 46 of 297 reptiles. Among 22 snakes, specific antibodies against pathogenic serovars of three Leptospira species (L. interrogans, L. kirschneri and L. borgpetersenii) at titre levels from 1:50 to 1:400 were detected in 6 snakes. In 31 of 210 lizards, specific antibodies were found in titres from 1:50 to 1:1000 and, finally, among 65 turtles (terrapins and tortoises), 9 had specific antibodies at titre levels between 1:50 and 1:1600. Animals imported from non-EU countries showed significantly higher prevalence (25.0%; 95 confidence interval: 16.7–33.3%) than animals from EU member states (10.4%; confidence interval: 6.1–14.7%). Conclusions: Reptiles may be considered as potential reservoirs of L. interrogans sensu stricto. Origin of the animals is a risk factor for presence of leptospiral antibodies, especially in lizards. Special attention should be focused on animals from non-EU member states.